I am trying to measure fatty acid oxidation in cultured myotubes. I saw that in many studies people add carnitine to their buffer. I wonder why do they do that? Is there not enough carnitine in cells to support fatty acid oxidation without the exogenous addition? What kind of oxidation will you detect if you do not add carnitine and use just the fatty acid solution in your oxidation buffer?
I am using Oxygraph device that measures oxygen flow into the cells in a closed chamber.
Skeletal myocytes do not synthesize L-carnitine; thus the nutrient must be added during myocyte differentiation to permit the cells to sequester an intracellular storage pool, as occurs in vivo. Myocytes cultured in the presence of only minuscule levels of carnitine (coming from the serum) may lead to a state of carnitine deficiency, and thus the results of experiments may be affected.
Thanks! I thought that the cells would have enough endogenous L-carnitine from the culture medium and serum, but you are obviously right, I would need to supplement it in my experiments. Thanks again for your advice!
You may keep in mind that peroxysomal oxidation is also going on in your cells.
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How can distinguish experimentally between the mitochondrial and peroxysomal oxidation?
I agree with Dr. Lehmann regarding inclusion of L-carnitine in differentiation medium. We use 100uM L-Carnitine and it has made a rather significant impact on FA oxidation measurements using 14C-FA. However, we have not tried this using FA substrates in an O2 consumption system.
Distinguishing between mitochondrial and peroxisomal oxidation can be tricky. The gold standard for assessing peroxisomal oxidation would be to use a very long chain FA, such as lignoceric acid (C24:0). We use 14C-lignoceric acid to measure CO2 release, but haven't tried this substrate in an oximetry system. Theoretically it may work, but rates of VLCFA oxidation are much slower than other lipids so it may be difficult to get a high level of sensitivity.
In 14C-FA oxidation studies, measuring incomplete oxidation (acid soluble metabolites; ASMs) in the presence of ETC inhibitors has been used for decades as a proxy for peroxisomal oxidation. However, this is also tricky since isolated mito preps still exhibit substantial ASM production even when peroxisomes are not present (i.e. this protocol seems to overestimate peroxisomal contributions). In terms of an O2 consumption system, inhibition of ATP synthase (antimycin or rotenone) gives you an idea of 'non-mitochondrial respiration'. Peroxisomes may contribute to this rate, but I am not aware of any studies that firmly establish 1) whether this is true, and 2) what relative percentage you could attribute to peroxisomes.
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